Compositions and methods for inhibiting intercellular interactions

ABSTRACT

Disclosed are compositions and methods involving the use of PRG4 protein, also known as lubricin, to mechanically inhibit biological processes involving cell motility and adhesion. The methods and compositions may be used to develop a variety of specific therapies and compositions, often exploited through surgical procedures, where development of the pathology involves one or more of the following modes of action: 1) the passage of cells from one body compartment to another, 2) adherence of macrophages to substrates such as fibrin or exposed extra cellular matrix, 3) binding of platelets to fibrin, or 4) failure of function of the glycocalyx on exposed epithelial cell surfaces, e.g., within the vasculature. In these instances PRG4 glycoprotein adheres to extracellular matrix or cell surfaces and presents a glycol-surface of polysaccharide which blocks the mechanisms of cell motility, extravasation, or intravasation, inhibits sticking of macrophages and platelets, and/or serves as a substitute or mimic of native glycocalyx.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/039,608 filed May 26, 2017, which is a U.S. national stageapplication filed under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2014/067464 filed Nov. 25, 2014, which claimspriority to and the benefit of U.S. Provisional Patent Application No.61/908,959, filed Nov. 26, 2013, the contents of each of which areincorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to compositions and methods involving the use ofPRG4 protein, also known as lubricin, to mechanically inhibit biologicalprocesses involving cell motility and adhesion. Uses include inhibitingthe metastases of cancers; inhibiting formation of intravascularthrombosis which may obstruct blood vessels and result in such events asa stroke, myocardial infarction, pulmonary embolism or the blockage ofblood vessels to other parts of the body; inhibiting the formation offibroses in organs or tissues and at the site of surgical incisions; andimproving circulation by protecting exposed endothelium from thedevelopment of atherosclerotic plaques, inhibiting the disruption ofpreviously formed plaques, inhibiting restenosis, and reducing shearstress in the vasculature.

BACKGROUND

The proteoglycan 4 gene (PRG4) encodes megakaryocyte stimulating factor(MSF) as well as a highly glycosylated different splice variant andglycoforms of “superficial zone protein” also known as lubricin.Superficial zone protein was first localized at the surface of explantcartilage from the superficial zone and identified in conditionedmedium. Lubricin was first isolated from synovial fluid and demonstratedlubricating ability in vitro similar to synovial fluid at acartilage-glass interface and in a latex-glass interface. It was lateridentified as a product of synovial fibroblasts, and its lubricatingability was discovered to be dependent on O-linked β (1-3) Gal-GalNAcoligosaccharides within a large mucin like domain of 940 amino acidsencoded by exon 6. Lubricin molecules are differentially glycosylatedand several naturally occurring splice variants have been reported. Theyare collectively referred to herein as PRG4. PRG4 has been shown to bepresent inside the body at the surface of synovium, tendon, articularcartilage such as meniscus, and in the protective film of the eye, amongother sites and plays an important role in joint lubrication andsynovial homeostasis.

Applicants have determined that beyond its ability to lubricate joints,tendons and cartilage, and the surface of the eye, lubricin may beuseful as a therapeutic to treat, prevent, or ameliorate a variety ofconditions where the utility of lubricin had not previously beenappreciated.

SUMMARY OF THE INVENTION

It has now been discovered that full length lubricin, or PRG4, ispresent in small concentrations in serum, and can act as a mechanicalblockade molecule to physically inhibit cellular binding involved in anumber of pathologic biochemical processes. These include metastasis,plaque formation, and thrombus formation. PRG4 capably interruptsplatelet and macrophage aggregation when added to a fibrin surface.While the mechanism of its effect has not yet been fully elucidated ordeeply examined, the inventors hereof speculate that PRG4 binds tosubstrates normally bound by vitronectin. As such, PRG4 functions notonly as a vitronectin binding inhibitor, but also as a protectivecoating which binds to exposed ECM and cell surfaces (polymorphonucleargranulocytes) with its heavily glycosylated central domain exposed, andacting as a surface lubricant and masking agent. In these embodiments,it functions as a glycocalyx, inhibiting native cell-cell andcell-matrix interaction.

Vitronectin is an abundant secreted glycoprotein found in serum andextracellular matrix. It binds to cell surfaces, heparin, chondroitinsulfate, collagen, and extracellular matrix (ECM), among otherbiomolecules, and promotes cell adhesion and motility. It has beenspeculated to be involved in hemostasis and tumor malignancy. Itcomprises three domains: an N-terminal somatomedin B domain, a centraldomain with hemopexin homology, and a C-terminal domain also withhemopexin homology.

The current invention uses applications of the PRG4 protein,appropriately glycosylated, preferably manufactured by expression of thePRG4 gene in a host cell such as Chinese hamster ovary, to mechanicallyinterrupt binding, motility and aggregation of immune, neoplastic orcancer cells. Coatings of PRG4 on physiological surfaces accordinglyinterrupt a number of pathological processes, and PRG4 can therefore beused in a number of novel ways in therapeutic and prophylactic contexts.

Thus, the invention encompasses methods for mitigating thrombosis andimproving circulatory function and health. It also provides ways toinhibit metastasis through boundary layer or film formation which in aload bearing situation is exemplified as boundary lubrication. It caninterrupt motility of cancer cells from solid tumors to the vasculatureand from the vasculature into the periphery. It also slows or inhibitsrestenosis of blood vessels after intravascular surgical procedures. Theinvention also provides methods for reducing or eliminating transplantrejection. The present invention further provides methods to reducedevelopment of fibroses following surgery, e.g., may be used to inhibitformation of scar tissue and fibrotic growth in surgically createdholes, grooves, and incisions, in contexts where a surgical incision orpassage is intended to remain open post-surgery. Among many examples, itmay be used in trabeculoplasty to maintain and improving flow of ocularfluids from the eye.

In one specific aspect, the invention provides compositions and methodsto reduce, inhibit or prevent unwanted migration of cells into or out ofa compartment of the body through the application of a coating rich inan exogenous PRG4 boundary lubricant onto a surface of a bodycompartment. Fundamentally, PRG4 mechanically inhibits cell-cell andcell-matrix interaction, thereby providing a method of inhibitingcell-cell adhesion. PRG4 can inhibit in a mammal transient bindinginteractions between a motile cell and a tissue. Accordingly, in oneaspect the invention comprises applying PRG4 to the surface of themotile cell or of the tissue in an amount sufficient to provide acoating which inhibits transient attractive interactions between thecell and the tissue so as to inhibit motility of the cell through,within, or across the surface of the tissue. The cell may be aneoplastic cell, a non-malignant neoplastic cell, a cancer cell, afibroblast, a macrophage, a neutrophil, eosinophil, basophil,lymphocyte, or a monocyte.

In one embodiment, the invention provides a method for reducing orinhibiting extravasation of cells from the circulatory system of apatient in need thereof by administering by injection directly orindirectly to the vascular system a composition of a physiologicallycompatible vehicle and PRG4 in an amount sufficient to contact PRG4 withan activated epithelial cell surface of the vasculature to produce aPRG4 film associated with at least a portion of the surface. As aresult, a boundary lubricant layer effective at inhibiting or reducingthe process of extravasation is provided.

In one embodiment, the cells are leukocytes or neoplastic cells whereasin another embodiment the cells are cancer cells, for example, prostate,breast, lung, or ovarian cancer cells. In another embodiment, the amountof PRG4 administered provides a dose of 0.3 mg/kg to 3.0 mg/kg of PRG4in the patient. According to the method, administration is by direct orindirect injection into the patient. Injection may involve intravenousinjection to provide local or systemic administration. Injection mayoccur into a body compartment of a patient, for example, the circulatoryor vascular system of a patient.

According to one embodiment, the inhibited or reduced extravasationevent is one step in the metastasis of a cancer. For example, metastasisto bone, lymph, lung, brain or liver of cancer cells is inhibited orreduced. According to another embodiment, administration of PRG4 occursin conjunction with surgical, chemotherapeutic, or radiologicaltreatment of a cancer, for example, to inhibit remaining cancer cellsfrom metastasizing to other areas of the body.

In another embodiment, the invention provides a method for reducing orinhibiting intravasation of cells from an extravascular compartment tothe circulatory system of a patient in need thereof by administering byinjection directly or indirectly to the extravascular compartment acomposition comprising a physiologically compatible vehicle and PRG4 inan amount sufficient to contact PRG4 with the cells or cell-surroundingmatrix to produce a PRG4 film associated with the surface of the cellsor surrounding matrix. As a result, a boundary lubricant layer effectiveat inhibiting or reducing the native process of cell motility orintravasation is provided.

In one embodiment, the cells are circulating neoplastic cells whereas inanother embodiment the cells are cancer cells, for example, prostate,breast, lung, or ovarian cancer cells. In another embodiment, the amountof PRG4 administered provides a dose of 0.3 mg/kg to 3.0 mg/kg of PRG4in the patient. According to the method, administration is by direct orindirect injection to the patient. Injection may involve intravenousinjection to provide local or systemic administration. Injection mayoccur into an extravascular compartment of a patient, for example, theintraperitoneal space, a tissue, or an organ or at the site of a tumoror at the site of a potential metastasis. In one embodiment, theinhibited or reduced intravasation event is the metastasis of a primarytumor to the circulatory or lymphatic system.

In still another aspect the invention provides methods of maintaining orimproving circulatory system function and health by administering byinjection directly or indirectly to the circulatory system a maintenanceamount of a composition comprising PRG4 and a physiologically compatiblevehicle. The amount is sufficient to deliver a concentration sufficientto form a boundary lubricating film of PRG4 at a stressed endothelialsurface or plaque within the vascular system.

In one embodiment of this method, the amount of PRG4 administered issufficient to provide a concentration in the patient of between 0.3mg/kg and 3.0 mg/kg. In another embodiment, the injection is a systemicintravenous injection or is a localized injection at the site of thestressed endothelial surface or plaque with the vascular system. In yetanother embodiment, the PRG4 improves circulatory system function byreducing flow instability within the vascular system.

According to another embodiment, the invention provides methods ofinhibiting or reducing vascular thrombus formation by administering byinjection directly or indirectly to the circulatory system of a patienta maintenance amount of a composition comprising PRG4 and aphysiologically compatible vehicle. The amount is sufficient to delivera concentration sufficient to form a boundary lubrication film of PRG4on fibrin and/or platelets present therein.

In one embodiment of this method, the amount of PRG4 administered issufficient to provide a concentration in the patient of between 0.3mg/kg and 3.0 mg/kg. In another embodiment, the injection is a systemicintravenous injection or is a localized injection at the site of athrombosis or at a site at risk of developing a thrombosis. For example,the site may be the site of an injury or tissue damage where clotformation is possible, the site of a developing deep vein thrombosis, orin the lung to prevent or reduce formation of pulmonary embolism.

In yet another embodiment, the invention provides a method of inhibitingvascular restenosis after an intravascular procedure by administering byinjection to the circulatory system of a patient systemically or locallyat the site of the procedure a composition comprising PRG4 and aphysiologically compatible vehicle. PRG4 is provided in an amountsufficient to provide a concentration sufficient to form a boundarylubricating film of PRG4 at an endothelial surface at the site withinthe vascular system.

In another embodiment, the intravascular procedure is a balloonangioplasty or a stent insertion. In yet another embodiment, the amountof PRG4 administered is sufficient to provide a concentration in thepatient of between 0.3 mg/kg and 3.0 mg/kg.

In yet another aspect, the invention provides a method of treating asurgical incision in a patient to maintain patency of the incision andprevent or limit fibrosis at the site of the incision comprisingadministering PRG4 in a physiologically compatible vehicle to the siteof the incision, whereby the lubricin prevents adherence of macrophagesto the site of the surgical incision, thereby preventing or limitingfibrosis at the site. According to one embodiment, the PRG4 isadministered at a concentration in a vehicle sufficient to provide acoating at a tissue surface, preferably of close packed lubricinmolecules noncovalently adhered to the ECM and/or cells of the tissuewith their lubricating domains directed upwardly from the tissuesurface.

In one embodiment, the surgical incision is in the trabecular meshworkof the eye. Accordingly, the invention provides methods of treating apatient suffering from ocular hypertension or glaucoma to improve thepatency and function of a surgically introduced passage through thetrabecular framework of the eye. The method comprises applying tosurfaces at the surgical site through or near the trabecular meshwork acomposition comprising PRG4 in a physiologically compatible vehicle at aconcentration sufficient to enhance flow of aqueous humor through thesurgically introduced passage. The method reduces the instance offibrotic clogging and occlusion at the site of the passage in thetrabecular meshwork.

In a further embodiment, the invention provides a method of treating,preventing or slowing the progress of fibrosis of an organ or tissuecomprising administering, preferably topically, to a patient sufferingfrom or at risk of fibrosis PRG4 in a physiologically compatible vehicledirectly or indirectly to the site at risk of fibrosis, whereby the PRG4inhibits adherence of macrophages at the site, thereby preventing orslowing the progression of fibrosis.

In yet another embodiment, the invention provides a method of inhibitingtransplant rejection comprising administering to the exterior surface ofa transplanted organ or tissue, and/or to the surface of a bodycompartment containing a transplanted organ or tissue, and/or to thevasculature of a patient having a transplanted organ or tissue acomposition comprising a physiologically compatible vehicle and PRG4 ata concentration sufficient to contact a cell surface with PRG4 toproduce a PRG4 film associated with the surface thereby to present aboundary lubricant layer effective at inhibiting normal cell-cellinteraction and to inhibit rejection of said transplanted organ ortissue. According to the invention, the transplanted organ or tissue maybe but is not limited to liver, kidney, heart, lung, eye tissue, tendon,or skin.

In any of the embodiments of this invention the patient may be a humanpatient.

In a further embodiment, the invention is directed to equipment forprocessing blood, specifically, the portions or compartments of theequipment that contact blood, is coated with lubricin. The equipment mayinclude dialysis equipment, or a heart-lung machine or equipment forprocessing blood transfusions and blood samples. In yet anotherembodiment, containers for holding and processing blood samples, such asvials, IV bags, or other containers are coated with PRG4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon depicting the interaction between endothelial cellsin the vascular endothelial bed 101, the approximately 500 nm in depth,highly charged glycocalyx 102, and immune cells 103 traversing along theendothelium, sampling the surface as it rolls. When peripheralinflammation is present 104, whether acute, chronic or auto-immune, acytokine gradient 105 signals certain endothelial cells to down regulateor cleave the protective glycocalyx 102, exposing surface receptors andhydrophobic binding sites, allowing immune cells to adhere, thenextravasate, migrating as shown by arrow 106 down the gradient. Thepresence of a suitable, spontaneously adherent boundary lubricant 107,e.g., PRG4 or lubricin, provides an abhesive, i.e., anti-adhesivesurface of exposed glycosylation and the immune cell is no longer ableto adhere or activate. This thereby interrupts the flow of cells out ofthe vasculature (extravasation). The same fundamental mechanism applieswhen PRG4 is used in an extravascular compartment which has developed asolid tumor, but the PRG4 acts instead to inhibit shed tumor cells fromthe cancer from migrating out of the compartment, or across a vascularor tissue interface. In essence, lubricin acts as a mechanical barrier,preventing cell attachment and subsequent motility.

FIG. 2A shows pictures taken with a bright field microscope of platescoated with fibrin and platelets (Panel 201—after 24 hours; Panel202—after 48 hours) and plates coated with fibrin, platelets, and PRG4(Panel 203—after 24 hours; Panel 204—after 48 hours). Dark spotsrepresent non-adhered platelets.

FIG. 2B is a bar graph quantifying the effect of lubricin on plateletadhesion in the plates shown in FIG. 2A. As shown, the level of plateletrecovery (i.e., non-adhered platelets) was dramatically higher fromsamples where lubricin was present.

FIGS. 3A-C show clotting of blood in CLOTMASTER™ Hula Cups of untreatedblood (FIG. 3A), blood treated with phosphate buffered saline (PBS; FIG.3B), and blood treated with recombinant human PRG4 (rhPRG4; FIG. 3C).While a clot formed in untreated blood and blood treated with PBS, bloodtreated with rhPRG4 resists clotting and flows freely in the bottom ofthe Hula Cup.

FIGS. 4A-B are line graphs presenting data showing the fibrokinetics ofsamples of normal human plasma (NHP; FIG. 4A) and pathological plasma(path plasma; FIG. 4B) pooled from liver disease patients as measured byoptical density over time. In FIG. 4A, the plotted data curve lines aretop to bottom 0 mg/mL lubricin (x), 50 mg/mL lubricin (▴), 100 mg/mLlubricin (▪) and 200 mg/mL (♦) lubricin. In FIG. 4B, the plotted datacurve lines are top to bottom 50 mg/mL lubricin (▴), 100 mg/mL lubricin(▪), 0 mg/mL lubricin (x), and 200 mg/mL (♦) lubricin.

FIGS. 5A-B are bar graphs presenting data demonstrating the ability ofPRG4 to interrupt macrophage adhesion to a fibrin surface underconditions similar to those described for the experiment in FIGS. 2A-B.Essentially no macrophages are able to bind to the fibrin surface whenPRG4 is added to the plate, with cell recoveries near 100% at 24, 48 and72 hours following exposure. This supports the hypothesis that PRG4 hasa long-lasting and potent ability to interrupt interactions of cells andmatrix. FIG. 5C provides cell count data recording the percentages offree macrophages recovered from a fibrin surface with and without PRG4.Because the effect is mechanical in nature, PRG4 protects the surfaceequally well against adhesion of a variety of other circulating celltypes, including cancer cells.

FIG. 6 is the amino acid sequence of full length (non-truncated) humanPRG4 (SEQ ID NO:1; 1404 residues). Residues 1-24 (shown in bold)represent the signal sequence and residues 25-1404 represent the maturesequence of human PRG4. The glycoprotein does not require the leadsequence in its active form.

FIG. 7 is the nucleic acid sequence for the PRG4 gene (SEQ ID NO:2)encoding the full length 1404 AA human PRG4 protein.

DETAILED DESCRIPTION OF THE INVENTION

The function of PRG4 heretofore has been almost entirely associated withprevention of wear between articulating joints and lubrication ofinterfacing tissues such as in the between the cornea and conjunctiva ofthe eye. Use of a systemic boundary lubricant such as PRG4 protein forthe purposes and uses described in the present invention to applicants'knowledge have not been previously suggested, possibly because the majorcomponent of the vascular endothelial glycocalyx (syndecans, glypicans,perlecans, versicans, decorin, biglycan, and mimecan, along with GAGssuch as heparan sulfate, chondroitin sulfate, dermatan sulfate, andhyaluronic acid reported in the literature (e.g., Weinbaum S, Tarbell JM, Damiano E R, Annu. Rev. Biomed. Eng. 2007. 9:121-67 & PflugersArch—Eur J Physiol (2007) 454:345-359)) do not include PRG4.

The functional importance of PRG4 in joint maintenance has been shown bymutations that cause the camptodactyly-arthropathy-coxavara-pericarditis (CACP) disease syndrome in humans. CACP is manifest bycamptodactyly, noninflammatory arthropathy, and hypertrophic synovitis,with coxa vara deformity, pericarditis, and pleural effusion. Also, inPRG4-null mice, cartilage deterioration and subsequent joint failurewere observed. Therefore, PRG4 expression is a necessary component ofhealthy synovial joints.

For the invention claimed herein, reduction in metastases is achieved bysupplementation of local (within a specific body compartment) orsystemic (within the vasculature) boundary lubrication using PRG4. PRG4binds to endothelial surfaces and may be used as a device or agent invivo to inhibit cell motility mechanisms, and prevent the unwantedmigration of cells, e.g., across surfaces, through tissues, or betweenphysiological compartments.

Physicochemical modes of lubrication broadly have been classified as“fluid film” or boundary. In biological systems, the operativelubrication modes depend on the normal and tangential forces on thearticulating tissues, on the relative rate of tangential motion betweenthese surfaces, and on the time history of both loading and motion. Thefriction coefficient, μ, provides a quantitative measure, and is definedas the ratio of tangential friction force to the normal force. One typeof fluid-mediated lubrication mode is hydrostatic. At the onset ofloading and typically for a prolonged duration, interstitial fluidbecomes pressurized, due to the biphasic nature of tissue; fluid mayalso be forced into the asperities between articular surfaces through aweeping mechanism. Pressurized interstitial fluid and trapped lubricantpools may therefore contribute significantly to the bearing of normalload with little resistance to shear force, facilitating a low μ. Also,at the onset of loading and/or motion, squeeze film, hydrodynamic, andelasto-hydrodynamic types of fluid film lubrication occur, withpressurization, motion, and deformation acting to drive viscouslubricant from and/or through the gap between two surfaces in relativemotion.

In some instances, the relevant extent to which fluid pressure/filmversus boundary lubrication occurs depends on a number of factors. Whenlubricant film can flow between the conforming sliding surfaces, whichcan deform elastically, elasto-hydrodynamic lubrication occurs.Pressure, surface roughness, and relative sliding velocity determinewhen full fluid lubrication begins to break down and the lubricationenters new regimes. As velocity decreases further, lubricant filmsadherent to the articulating surfaces begin to contribute and a mixedregime of lubrication occurs. If the velocity decreases even further andonly an ultra-thin lubricant layer composed of a few molecules remain,boundary lubrication occurs. In certain instances, the boundary mode oflubrication is therefore indicated by a friction coefficient duringsteady sliding being invariant with factors that influence formation ofa fluid film, such as relative sliding velocity and axial load. Forcertain tissues in the body, such as articular cartilage, it has beenconcluded that boundary lubrication occurs, and is complemented by fluidpressurization and other mechanisms. In traditional boundary lubricationtheory, increasing loading time and dissipation of hydrostatic pressureallows lubricant-coated surfaces to bear an increasingly higher portionof the load relative to pressurized fluid, and consequently, this modecan become increasingly dominant.

The inventors hereof have recognized that the velocity profile of bloodand lymph flowing through the vasculature approximates the no-slipcondition used in fluid mechanics to describe a radially dependentreduction in velocity and concurrent increase in blood pressure near thewalls of a vessel to create the ideal conditions for boundarylubrication. It is also recognized that surface bound glycoproteins arelargely responsible for the transmission of shear stress to theendothelial cells, and that mechanotransduction of that shear isnecessary for proper regulation of cell morphology, nitric oxide (NO)production, cytoskeletal reorganization and hyaluronan content (PflugersArch—Eur J Physiol (2007) 454:345-359). In particular, areas of lowshear, unstable and oscillatory flow are prone to atherosclerosis,impaired NO production and an increase in hyaluronidase. Similarly,improvement in shear transmission is associated with an increase inglycocalyx thickness and hyaluronan content. Laminar flows within normaltissue result in an approximately 400 nm glycocalyx thickness, whileregions exposed to disturbed flow exhibited thinner coatings of lessthan 100 nanometers (van den Berg B M, et al. (2006) Am J Physiol HeartCirc Physiol 290:H915-H920).

Accordingly, the present invention, among other aspects, contemplatesthe application of recombinant, exogenous surface binding lubricantPRG4, typically by introduction into the vasculature, to: 1) provideincreased boundary lubrication (and steric masking of underlyingreceptors, integrins and selectins, etc.) between cells and cellinterfaces to prevent or substantially reduce extravasation of cellsbetween compartments (e.g., rolling, adhesion,transmigration/diapedesis); 2) bind to endogenous exposed endothelialcells to improve the transmission of shear to the underlying cells(e.g., improved mechanotransduction, improved nitric oxide production,improved production of endogenous glycocalyx); 3) extend the glycocalyxfurther into the lumen to smooth out the flow, reduce oscillations,instabilities, and improve laminar flow, and 4) replace, mask or competewith endogenous aberrant PRG4 so as to prevent binding to L-selectin onpolymorphonuclear granulocytes.

As disclosed herein, PRG4 may provide boundary lubrication along thewalls of the vasculature. In some embodiments, it protects peripheraltissues from extravasation of cells such as cancer cells, helps totransmit shear stress to underlying endothelium, and reduces surfaceroughness to reduce fluid instability.

The methods and compositions may be used to develop a variety ofspecific therapies and compositions, often exploited through surgicalprocedures, where development of the pathology involves one or more ofthe following modes of action: 1) the passage of cells from one bodycompartment to another, 2) adherence of macrophages to exposedextracellular matrix or fibrin 3) binding of platelets to fibrin, or 4)failure of function of the glycocalyx on exposed epithelial cellsurfaces, e.g., within the vasculature. In these instances PRG4 proteinadheres to extracellular matrix or cell surfaces and presents aglycosylated surface of short polysaccharide chains which blocks themechanisms of cell motility, extravasation, or intravasation, inhibitssticking of platelets, and/or serves as a substitute or mimic of nativeglycocalyx. As examples, PRG4 may be used to inhibit metastases ofcancers, thereby preventing unwanted migration of cancer cells into orout of a physiological compartment of the body. As another example, PRG4may be exploited to mechanically interrupt binding, motility andaggregation of immune cells, inhibit formation of intravascularthrombosis. Most generally, the invention achieves these effects throughthe direct or indirect application to tissue surfaces of exogenous PRG4protein, which behaves in vivo as a boundary lubricant.

For the invention claimed herein, reduction in metastases is achieved bysupplementation of local (within a specific body compartment) orsystemic (within the vasculature) boundary lubrication using PRG4. PRG4binds to endothelial surfaces and may be used as a device or agent invivo to inhibit cell motility mechanisms, and prevent the unwantedmigration of cells, e.g., between physiological compartments.

As used herein, “body compartment” or “physiological compartment” refersto any locality that is physically separated by a tissue from others andhouses a tissue or system of interest. In the case of a solid tumor, thecompartment is the tissue immediately surrounding the tumor boundary. Inthe case of a surgically applied incision, e.g., during minimallyinvasive glaucoma surgery, it would comprise the void left by theincision. In the case of closed-angle glaucoma surgery, the compartmentis defined as the anterior chamber. In the case of preventing transplantrejection, the compartment would be the tissue surrounding thetransplant. In the case of inhibiting restenosis, the compartment wouldbe the inside of the vessels that receives the stent or other surgicalinsult.

In order for cancer cells to successfully colonize a metastatic site,they must detach from the primary tumor using extracellularmatrix-degrading proteases, intravasate and survive in the circulation,evade the immune response, and extravasate the vasculature to invade thetarget tissue parenchyma, where metastatic foci are established. Themethods of invention can be used to prevent or reduce the incidence ofintravasation, i.e., movement of cells from the tissues into the bloodor lymph. With respect to these cells, the methods of the invention canalso be used to prevent or reduce the incidence of extravasation, i.e.,movement of cells from the blood into the tissues and organs of thebody. In particular, the methods of the invention can be used to preventmetastases to the lymph, lungs, liver, brain, and/or bones of cancer andneoplastic cells. These are the most common body tissues for metastasisby primary tumors, although the methods of the invention can be used toprevent or reduce metastasis to other organs and tissues.

The Active PRG4 Moiety

Lubricin is a lubricating polypeptide, which in humans is expressed fromthe megakaryocyte stimulating factor (MSF) gene, also known as PGR4 (seeNCBI Accession Number AK131434-U70136). Lubricin is a ubiquitous,endogenous glycoprotein that coats the articulating surfaces of the body[Jay G D 2004]. Lubricin is highly surface active molecule (e.g., holdsonto water), that acts primarily as a potent cytoprotective,anti-adhesive and boundary lubricant. It is characterized by a long,central mucin-like domain located between terminal protein domains thatallow the molecule to adhere and protect tissue surfaces. Its naturalform, in all mammals investigated, contains multiple repeats of an aminoacid sequence which is at least 50% identical to KEPAPTT. Naturallubricin typically comprises multiple redundant forms of this repeat,but typically includes proline and threonine residues, with at least onethreonine being glycosylated in most repeats. The threonine anchoredO-linked sugar side chains are critical for lubricin's boundarylubricating function. The side chain moiety typically is aβ(1-3)Gal-GalNAc moiety, with the β(1-3)Gal-GalNAc typically capped withsialic acid or N-acetylneuraminic acid [Jay G D 2001]. The polypeptidealso contains N-linked oligosaccharides. The gene encodingnaturally-occurring full length lubricin contains 12 exons, and thenaturally-occurring MSF gene product contains 1,404 amino acids withmultiple polypeptide sequence homologies to vitronectin includinghemopexin-like and somatomedin-like regions. Centrally-located exon 6contains 940 residues. Exon 6 encodes the repeat rich, O-glycosylatedmucin domain.

The amino acid sequence of the protein backbone of a lubricatingpolypeptide may differ depending on alternative splicing of exons of thehuman MSF gene. Because lubricin serves a fundamentally mechanicalfunction, its fine tertiary structure is less critical than proteinssuch as cytokines or antibodies which depend on subtle stereochemistrywhich governs binding to receptors. This robustness againstheterogeneity was exemplified when researchers created a recombinantform of lubricin missing 474 amino acids from the central mucin domain,yet still achieved reasonable, although muted, lubrication [Flannery C R2009]. PRG4 has been shown to exist not only as a monomer but also as adimer and multimer disulfide-bonded through the conserved cysteine-richdomains at both N- and C-termini [Schmidt T A 2009]. Lμbris, LLC hasdeveloped a full-length recombinant form of human lubricin. The moleculeis expressed using the Selexis Chinese hamster ovary cell line (CHO-M),with a final apparent molecular weight of 450-600 kDa, with polydispersemultimers frequently measuring at 2,000 kDa or more, all as estimated bycomparison to molecular weight standards on SDS tris-acetate 3-8%polyacrylamide gels. Of the total glycosylations, about half of themolecule contain two sugar units (GalNAc-Gal), and half three sugarunits (GalNAc-Gal-Sialic acid.

Any one or more of various native and recombinant PRG4 proteins andisoforms may be utilized in the various embodiments described herein.For instance, U.S. Pat. Nos. 6,433,142; 6,743,774; 6,960,562; 7,030,223,and 7,361,738 disclose how to make various forms of human PRG4expression product, each of which is incorporated herein by reference.Preferred for use in the practice of the invention is full length,glycosylated, recombinant PRG4, or lubricin, expressed from CHO cells.This protein comprises 1404 amino acids (see FIG. 6; SEQ ID NO:1)including a central exon comprising repeats of the sequence KEPAPTTvariously glycosylated with O-linked β (1-3) Gal-GalNAcoligosaccharides, and including N and C-terminal sequences with homologyto vitronectin. The molecule is polydisperse with the glycosylationpattern of individual molecules varying, and can comprise monomeric,dimeric, and multimeric species.

As used herein, the term “PRG4” is used interchangeably with the term“lubricin.” Broadly, these terms refer to any functional isolated orpurified native or recombinant properly glycosylated PRG4 proteins,homologs, functional fragments, isoforms, and/or mutants thereof. Alluseful molecules comprise the sequence encoded by exon 6, or homologs ortruncated versions thereof, for example, versions with fewer repeatswithin this central mucin-like KEPAPTT-repeat domain, together withO-linked glycosylation. All useful molecules also comprise at least thebiological active portions of the sequences encoded by exons 1-5 and7-12, i.e., sequences responsible for imparting to the molecule itsaffinity for ECM and endothelial surfaces. In certain embodiments, apreferred PRG4 protein has an average molar mass of between 50 kDa and500 kDa, preferably between 224 to 467 kD, comprising one or morebiological active portions of the PRG4 protein, or functional fragments,such as a lubricating fragment, or a homolog thereof. In a morepreferred embodiment, a PRG4 protein comprises monomers of average molarmass of between 220 kDa to about 280 kDa.

Methods for isolation, purification, and recombinant expression of aPRG4 protein are well known in the art. In certain embodiments, themethod starts with cloning and isolating mRNA and cDNA encoding PRG4proteins or isoforms using standard molecular biology techniques, suchas PCR or RT-PCR. The isolated cDNA encoding the PRG4 protein or isoformis then cloned into an expression vector, and expressed in a host cellfor producing recombinant PRG4 protein, and isolated from the cellculture supernatant.

Particular forms of PGR4 constructs may readily be tested for theirability to inhibit chemotaxis and cell motility effectively ex vivo. Awide variety of qualitative and quantitative assay techniques are knownwhich permit determination of whether cell motility is interrupted andto measure the intensity of the responses. See, for example, the seminalwork of Boyden, The Chemotactic Effect Of Mixtures Of Antibody AndAntigen On Polymorphonuclear Leucocytes, J Exp Med. 1962 Feb. 28;115(3): 453-466.

For use in the practice of the invention PRG4 may be formulated in acarrier, e.g., suspended in phosphate buffered saline, at concentrationsranging from 1 μg/mL to 10 mg/mL, and more preferably, 100-500 μg/mL.Depending on the specific use, PRG4 is administered parenterally e.g.,by injection, daily, weekly, or monthly. It may be administered forsystemic distribution through the vasculature, or locally within thevasculature during the course of vascular surgery such as balloonangioplasty and/or stent insertion. It may be injected into a bodycompartment, e.g., during endoscopic or open wound surgery. Its effectin each case is to bind to cellular or ECM surfaces resulting ininhibition of cell motility and chemotaxis. In yet other embodiments,PRG4 is combined with other components of a boundary lubrication systemsuch as hyaluronic acid (HA), anti-coagulants such as heparin, GAGs suchas chondroitin sulfate or heparan sulfate, or TIMPs (Tissue Inhibitor ofMetalloproteinase), preferably TIMP1, TIMP2, TIMP3, or TIMP4.

Such PRG4 compositions may be used in the following ways to achieve thefollowing effects.

Inhibition of Atherosclerosis

A variety of studies have revealed that atherosclerosis is found inregions of low shear stress within the arterial vasculature. In suchlocations, a reduction in endothelial nitric oxide synthase production,and concomitant upregulation of VCAM-1 adhesion factors leads to anincrease in the adhesion of monocytes in atherosclerotic plaques. (ChengC., et al. Cell Biochemistry and Biophysics, 2004; 41:279-294). Thisincrease may be traced to an accumulation of macrophages. The presentembodiment takes advantage of the ability of PRG4 to inhibit and indeedto almost entirely prevent macrophage accumulation (FIGS. 5A-C), therebyto interrupt the formation and growth of atherosclerotic plaques, andfurther to reduce the likelihood of plaque rupture.

In the present embodiment, PRG4 injected throughout the vasculature alsobinds up- and downstream from the plaque, where macrophages tend toaccumulate, masking adhesion factors, reducing flow instabilities,preventing particle aggregation and therefore preventing furthermacrophage association through these factors in addition to its abhesiveproperties.

Mitigation of Restenosis and Thrombosis in Stent Surgery and BalloonAngioplasty

Restenosis is caused in part by low shear stress that tends to encouragemacrophage accumulation. In turn, macrophages mediate dissolution of theelastic membrane, and eventually lead to neointimal hyperplasia,increased expression of vascular cell adhesion molecule-1, and decreasednitric oxide production. In contrast, higher shear stress reducesmacrophage accumulation and elastic membrane proteolysis (Carlier S G,et al. Circulation 2003; 107:2741-2746). Accordingly, in one embodimentof the invention, PRG4 injected into the circulatory system or irrigatedinto the intravascular surgical site during surgery, locally binds tothe endothelium and exposed extracellular matrix. This serves to inhibitor prevent macrophage accumulation, improve shear mechanotransductionthrough a “reconstituted” glycocalyx, and to restore proper endothelialgene expression with the result that restenosis is inhibitedsignificantly.

Mitigation of Metastasis

In the first order approximation, metastatic tumor cells requirefunctioning cell motility mechanisms to achieve migration and invasion.Cancer cell migration is similar to normal migration, although in a morerandom fashion. The metastatic cell extends filopodia or broadlamellipodia, which adhere to extracellular matrix or to adjacent cellsthrough transmembrane receptors to the actin cytoskeleton, upon whichthe cell pulls itself in the direction of the adhered protrusion, or asdescribed by Lauffenburger D A et al., motility requires morphologicalpolarization, membrane extension, formation of cell-substratumattachments, contractile force and traction, and release of attachments(Lauffenburger D A, et al. Cell 1996; 84(3):359-369 and Hongyu Z, CritRev Eukaryot Gene Expr. 2010; 20(1): 1-16). Essentially, cancer cellscrawl by extending adherent fingers and pulling themselves forward.While typically it is believed that only malignant tumors metastasize,it is now thought that non-malignant tumors may also be capable ofmetastasis. Accordingly, the method is applicable to both malignant andnon-malignant neoplastic cells. According to the invention, PRG4, apotent boundary lubricant is injected to the periphery in the spacewhere a tumor has been excised, or around an otherwise inoperable orlocated solid tumor, or within the circulatory system to coat thecellular interface and prevent cancer cells from forming cell-substratumattachments. According to this embodiment, the objective is to providePRG4 as a motility inhibitor such that the tumor cells cannot attach,cannot crawl, and therefore cannot achieve migration or invasion. It isalso recognized that tumors typically engage in extensive intravasationand extravasation, followed by growth and angiogenesis to repeat theprocess of metastasis. In the current embodiment, systemic or locallyapplied PRG4 is used to prevent intravasation or extravasation of tumorcells.

A series of studies also suggest that there is a strong associationbetween thrombosis and the progression of cancer, with some studiessuggesting that hijacking of the coagulation system may be critical forthe survival and spread of the tumor cells; in particular, fibrin andplatelets seem to prevent clearing of tumor cells by natural killercells (Palumbo J S, et al. Blood 2005; 105: 178-85, Bakewell S J, et al.PNAS 2003; 100:14205-14210, Lazo-Langer A, et al., Journal of Thrombosisand Haemostasis, 2007; 5:729-737). Clinical studies have alsodemonstrated that fibrinogen deficient animal models reduced theincidence of spontaneous macroscopic metastases in the lung and regionallymph nodes, although primary tumor growth and angiogenesis wereunaffected (Palumbo J S, et al., Cancer Res 2002; 62:6966-6972). It hasalso been hypothesized that tumor cells may use local platelet-fibrindepositions to support sustained adhesions of tumor cells within highshear environments, and provide a means for cell proliferation (PalumboJ S, et al. Blood 2005; 105: 178-85). The invention therefore makes useof the ability of PRG4 to almost entirely inhibit the associations ofplatelets and fibrin (FIGS. 2A-B, 3A-C) to interrupt the metastaticpotential of the circulating or solid tumor cells. In addition, thecurrent invention suggests that the boundary properties of a PRG4 coatedsurface may also shield selectin and integrin mediated plateletactivation that is critical to tumor invasion. In one preferredembodiment, PRG4 protein may be injected around the site of aninoperable solid tumor, e.g., a poorly located brain tumor or mass nearvital organs in the throat, to minimize metastatic potential of thetumor, and to prevent extravasation of tumor cells into the surroundingcompartments. In particular, the invention contemplates prevention orreduction in extravasation and metastasis of primary tumors to thelungs, lymph, liver, bone and brain as these are the most commonlocations of metastatic tumors.

For example, in one specific embodiment of the invention, lubricin isused to treat, alleviate, or prevent bone metastasis, also known asmetastatic bone disease, which results from invasion into the bone of aprimary tumor. Bone tissue is a common location for cancer metastasis.The primary tumor causing metastasis may be, for example, lung cancer,prostate cancer, or breast cancer. Treatment of bone metastasis,according to the invention, involves administration of lubricin to apatient suffering from or at risk of developing bone metastasis.Lubricin may be administered to a patient as a pretreatment, for exampleto prevent or reduce the likelihood of bone metastasis when a non-boneprimary tumor has been diagnosed in the patient. Lubricin may also beadministered to a patient to prevent, treat, or alleviate bonemetastasis in conjunction with administration of a chemotherapeuticagent or radiation treatment to treat a primary tumor or a metastaticcancer. According to one embodiment, the lubricin is administeredlocally to the bone by injection at the site of metastasis or potentialmetastasis or it is administered systemically to the patient. In oneembodiment, the patient is preferably a human patient.

Lubricin may also be used to treat or prevent ovarian cancer metastasis.It is believed that ovarian cancer metastasizes by seeding tumor cellsonto the mesothelial layer lining the peritoneum. Accordingly, in oneembodiment, lubricin is administered to a patient having ovarian cancer.In one embodiment, the lubricin is administered systemically, while inanother embodiment, the lubricin is administered to the peritoneum inthe region of an ovarian tumor in the patient. The patient, in oneembodiment, is a human patient.

A non-limiting of list of cancers for which metastasis may be preventedor reduced by administration of lubricin according to the inventioninclude adrenal cancer, anal cancer, bile duct cancer, bladder cancer,bone cancer, brain/CNS cancer, basal cell skin cancer, breast cancer,Castleman disease, cervical cancer, colorectal cancer, endometrialcancer, esophagus cancer, dermatofibrosarcoma protuberans, Ewing familyof tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoidtumors, gastrointestinal stromal tumor (GIST), gastric cancer,gestational trophoblastic disease, glioma, glioblastoma, head and neckcancer, hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal andhypopharyngeal cancer, leukemia, lung cancer, liver cancer, lymphoma,malignant mesothelioma, Merkel cell carcinoma, melanoma, multiplemyeloma, myeloma, myelodysplastic syndrome, nasal cavity and paranasalsinus cancer, nasopharyngeal cancer, neuroendocrine cancer,neuroblastoma, Non-Hodgkin lymphoma, oral cavity and oropharyngealcancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,pituitary Tumors, prostate cancer, renal cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma, squamous cell skincancer, small intestine cancer, stomach cancer, testicular cancer,thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma, vaginalcancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

Prevention of Fibrosis

Fibrosis occurs when fibroblasts produce connective tissue, i.e., scartissue, sometimes in excess, in an organ or tissue. Generally, fibrosisoccurs as a result of the reparative process. When a tissue is injured,for example, through surgical means or other physical injury,macrophages are recruited to the site of the injury and, along with thedamaged tissue, produce TGF-beta, which signals fibroblasts to produceconnective tissue to close the wound. In some cases, fibrosis occurs inthe absence of a surgical insult or physical injury to the tissue. Forexample, viral or bacterial infections or genetic abnormalities may leadto fibrosis. Many instances of fibrosis are idiopathic. Nonetheless,whether caused by physical injury or not, the invention provides thatfibrosis may be alleviated, prevented or slowed through the use of PRG4.Essentially, PRG4 provides a mechanical barrier to prevent or limitmacrophage accumulation at the site of tissue injury to prevent or limitfibrosis, or in the case of an organ or tissue that is already fibrotic,to limit or retard the progress of fibrosis by reducing or preventingthe accumulation of further macrophages at the site.

According to one embodiment, the invention provides a method forpreventing, alleviating or slowing fibrosis by administering to apatient at risk of developing fibrosis or experiencing fibrosis in anorgan or tissue an effective amount of PRG4 in a pharmaceutical carrier.The PRG4 may be administered to the patient directly or indirectly byinjection. For example, the PRG4 may be administered systemically to thevasculature of the patient or it may be administered locally ortopically to the site at risk of developing fibrosis or where fibrosisis present. The site of local administration may be an organ or tissueor the surface of the organ or tissue.

In some instances, as discussed above, fibrosis is not the result of asurgical or physical tissue injury, but rather is the result of anothercause such as infection, underlying genetic abnormalities, oridiopathic. Some examples of fibrosis that are not necessarily caused bysurgical insults, include cirrhosis of the liver, pulmonary fibrosis,cardial fibrosis, mediastinal fibrosis, arthrofibrosis, myelofibrosis,nephogenic systemic fibrosis, keloid fibrosis, scelorderma fibrosis,cystic fibrosis, renal fibrosis, lymphatic tissue fibrosis, arterial,capillary, and vascular fibrosis, and pancreatic fibrosis.

In another embodiment, the invention provides a method for preventing orslowing fibrosis at the site of a surgical incision in a patient inorder to maintain patency of the surgical incision by administering tothe patient an effective amount of PRG4 in a pharmaceutically acceptablecarrier. The PRG4 may be administered to the patient directly orindirectly by injection. For example, the PRG4 may be administeredsystemically to the vasculature of the patient or it may be administeredlocally or topically to the site of the surgical incision.

For example, the purpose of some surgeries is to provide an opening inthe tissue that does not close. This can be useful in order to providedrainage from a tissue or organ to relieve pressure or to remove fluid,to evacuate waste products, or to place a device that will eventually beremoved, i.e., a temporary device where it is not desirable for thedevice to “heal in.” Accordingly, in these instances, providing PRG4locally at the site of the surgical incision helps prevent or reduce theinstance of fibrosis at the site.

Surgical methods for providing an incision not intended to close includesurgically produced stomas such as dacryocystorhinostomy (a surgicallyformed connection between the nose and lacrimal sac); gastrointestinalstomas such as a cecostomy, colostomy, duodenostomy, ileostomy,jejunostomy; esophagostomy; gastrostomy; cholecystostomy;choledochostomy; sclerostomy; tracheostomy, urostomy, nephrostomy,ureterostomy, vesicostomy, cystostomy, and enterostomy; an ileoanalreservoir; a site for placement of a percutaneous catheter or stent; orsurgical windows (both exterior and interior) in the skull and sinuses.For example, the Caldwell-Luc operation relieves chronic sinusitis bycreating a “window” to connect the maxillary sinus with the nose, thusimproving drainage of the maxillary sinus, one of the cavities beneaththe eye.

Further, the invention provides methods to maintain separation oftissue, organs and/or tendons after an adhesiolysis or surgical release.Adhesions may form as the result of prior surgery or from otherconditions that permit scar tissue to form between tissues and/or organsthat are otherwise not connected. Adhesions may be surgically “lysed” orcut. Surgical release may be performed to release tendons and connectivetissue that are improperly attached or where the attachment otherwisecauses pain. Accordingly, the invention provides methods to preventreconnection of tissues, tendons and/or organs that are separated byadhesiolysis or surgical release by administering PRG4. The PRG4 may beadministered to the patient directly or indirectly by injection. Forexample, the PRG4 may be administered systemically to the vasculature ofthe patient or it may be administered locally or topically to the siteof the adhesiolysis or surgical release. In the case of carpal tunnelsyndrome, PRG4 may be administered to the site of a transection ofretinaculum.

Improvements in Trabeculoplasty or Trabeculectomy

The current embodiment provides for irrigation and or injection of PRG4to prevent restenosis of trabeculoplasty or trabeculectomy in the humaneye performed during an ocular surgery, for example, to treat glaucomaby relieving pressure in the eye. The incision in or removal of part ofthe trabecular mesh permits aqueous humor to drain from the eye,relieving pressure. However, the incision in the trabecular mesh may beprone to closure and fibrosis due to exposure of damaged epithelium andmigration of macrophages to the site to promote tissue repair.

Accordingly, one embodiment of this invention includes the use of PRG4during glaucoma surgery to prevent migration of macrophages and othercells involved in tissue repair to the incision. Accordingly, in onepreferred embodiment, the boundary lubricating and boundary film formingabilities of PRG4 coated tissues is used to effect a minimally invasivetrabeculoplasty or trabeculectomy. In one aspect of this method, ratherthan creating a fornix- or limbus-based conjunctival flap, or implantinga subchoroidal stent in higher risk patients, very small needles (e.g.,27 to 34 gauge) filled with PRG4 are passed through the trabecularmeshwork into the anterior chamber, followed by retraction with deliveryof the PRG4 to fill and coat the channel created by the syringe withboundary lubricant. By repeating this process in a radial fashion,minimal damage is done to the eye, no foreign substance is introducedinto the eye, drainage can be distributed radially, reducing the needfor blebs, additional channels can easily be added if more drainage isnecessary, and the PRG4 coated channels prevent restenosis.

Increase in Nitric Oxide Production

The current embodiment also provides for replenishing the vascularglycocalyx so to better transmit shear stress to the endothelial cellsbelow. This serves to generally enhance vascular health and maintenance,and to preserve endothelial NO production. One interesting sequelae ofpoor NO production is the inability to sustain a high quality orsustained erection. In particular, nitric oxide binds to guanylatecyclase receptors, upregulating cGMP, causing vasodilation, corpuscavernosal smooth muscle relaxation and eventually penile erection(Webb, D. J., et al. Am. J. Cardiol 1999; 83(5A): 21C-28C). In thecurrent embodiment, replenishment/enhancement of the vascularendothelial glycocalyx with PRG4 leads to better, more sustainederections. The current embodiment also envisions use of external,epithelial PRG4 to achieve complementary effects.

Mitigation of Transplant Rejection

Traditional immunosuppressive therapy, e.g., corticosteroids,calcineurin inhibitors, mTOR inhibitors, and anti-proliferativesfollowing tissue transplant surgery carries a variety of unfortunateside effects. One embodiment of the current invention uses periodicinjections of PRG4 around the transplanted tissue to create a mechanicalbarrier based on boundary film formation to dramatically reduce theincidence of hyperacute, acute and chronic rejection. PRG4 coatedsurfaces prevent cellular and humoral immune cells from binding to andrecognizing the transplant, but allows the immune system to functionnormally in the rest of the body. According to the invention, the methodmay be used to treat transplanted heart, liver, kidney, lung, skin ortendon or any other transplanted organ or tissue.

Inhibition of Thrombosis

PRG4 may be useful for inhibiting formation of intravascular thrombosiswhich may obstruct blood vessels, resulting in such events as stroke,myocardial infarction, pulmonary embolism, or blockage of blood vesselsto other parts of the body.

To demonstrate the anti-thrombotic effect of PRG4, the ability of PRG4to interrupt platelet adhesion to a fibrin surface was tested. Theresults are shown in FIGS. 2A-B.

Plastic cell culture dishes were coated with 10 μL of thrombin at 50U/mL and 120 μL fibrinogen at 270 mg/mL, suspended in 10 mL of water,and incubated for 3 hours at 37° C. After 3 hours, dishes were rinsedwith autoclaved deionized water. Next, 10 μL of PRG4 at 1.42 mg/mL in 10mL of water were added to the dish shown in panel 203 and 204 andincubated at 37° C. for 2 hours. Following the incubation, dishes werewashed again with autoclaved deionized water and 6×10⁵ platelets insterile PBS were added to each dish, mixed and incubated at roomtemperature.

Panel 201 shows a fibrin only dish at 24 hours, while panel 202 showsfibrin only dish at 48 hours; note that in both fibrin-only panels,there are few non-adhered platelets observable in the bulk solution(e.g., the small dark circles). Panel 203 shows a PRG4 and fibrin dishat 24 hours, while panel 204 shows PRG4 and fibrin dish at 48 hours,which have a significantly higher number of non-adhered platelets.

The effect of PRG4 on platelet adhesion (quantifying the recovery ofcells as a percentage of the original sample) is shown in FIG. 2B. Asillustrated, the exceptional recovery of platelets from PRG4 coateddishes indicates PRG4's ability to prevent platelets from binding to thefibrin surface.

In a second experiment, PRG4's ability to inhibit thrombogenesis wasobserved in vitro by testing clot formation in the presence and absenceof PRG4. Three samples of 30 mL of whole blood were obtained viavenipuncture and placed in CLOTMASTER™ Hula Cups (Pierce Surgical Corp.Stowe, Vt.) with a sintered glass core. To one sample 1.5 mL ofphosphate buffered saline (PBS) was added as a negative control to showthe result when no anticoagulant effect is expected; to the secondsample 1.5 mL rhPRG4 080 at a concentration of 1 mg/mL was added. Thethird sample was used as a control and was untreated. Each sample wasswirled for 60 seconds; swirling stopped and the containers remainedstatic for 9 minutes to promote clot formation. Clot quality was thenassessed and clots were decanted and placed in formalin. The results areshown in FIGS. 3 A-C where the photographs show that in the control clotformation was observed as can be seen on the sintered glass core and inthe bottom of the cup as a viscous mass (FIG. 3A). For the PBS sample,clot formation occurred as is evidenced by the viscous mass in thebottom of the cup (FIG. 3B). In contrast, no clot formation was observedin the presence of as the blood is still fluid and flowed to one side ofthe cup when tipped (FIG. 3C). This data strongly demonstrates theanti-thrombotic properties of PRG4.

In a third experiment, the effect of PRG4 on the fibrokinetic profile ofnormal and pathological plasma was tested. Normal human plasma (“NHP”)was supplemented with lubricin (2 mg/mL) at a 1:10 dilution. Subsequent1:2 and 1:4 dilutions were made in normal human plasma. Pooled plasmafrom liver disease patients (“Path plasma”) was also supplemented withlubricin (2 mg/mL) at a 1:10 dilution. Subsequent 1:2 and 1:4 dilutionswere made in pooled liver disease plasma. The formation of a fibrin clotwas tested by reading optical density at 405 nm for 30 minutes at 15second intervals on 200 μL plasma samples of each dilution mixed with 25μL CaCl₂ (0.25 mM) and 25 μL thrombin (5 U/mL). Samples with no addedlubricin (0 mg/mL) were used as controls. As shown in FIGS. 4A and 4B,lubricin at a concentration of 200 μg/mL decreased optical density inboth normal human plasma and pathologic plasmas which indicates that therate of fibrin (clot) formation was decreased in each sample (see bottomdata line in each of FIGS. 4A and 4B). These experiments suggests thatlubricin interferes with fibrin clot formation. Accordingly, this datasuggests that lubricin has antithrombotic properties.

The anti-thrombotic properties of PRG4 make it useful as ananticoagulant. For example, PRG4 may be used in diagnostic applicationsas an anticoagulant. For example, in one embodiment, PRG4 is used tocoat containers and vials designed for capturing or containing orprocessing a blood sample. In another embodiment, dialysis equipment,surgical equipment, stents or other devices for implantation in a humanor animal body are coated with lubricin to act as an anticoagulant.

In another embodiment, PRG4 is administered to a patient, for example,by injection or another administration method described herein as ananticoagulant therapy. For example, PRG4 may be administered to apatient to prevent or reduce clot formation in a patient suffering fromor at risk of thrombosis from deep vein thrombosis; pulmonary embolism;a clotting disorder such as Factor V Leiden, Prothrombin 20210 mutation,hyperhomocysteinemia, Protein C deficiency, Protein S deficiency,Antithrombin deficiency, Von Willebrand Factor disorder, or elevatedlevel of procoagulant protein such as VIII, IX, XI, VII, fibrinogen, andVon Willebrand factor; a heart condition such as atrial fibrillation,mechanical or prosthetic valve, or patent foramen ovale. Administrationof PRG4 prevents or reduces clot formation in such patients. PRG4 may beadministered systemically or locally to a site at risk of thrombosis orwhere a thrombosis is present. In these instances, PRG4 may beadministered to a patient in conjunction with another anticoagulanttherapy such as heparin, warfarin, coumarin, or dabigatran. PRG4 may beadministered as a prophylactic in patients with the aforementionedconditions to prevent clotting due to the patient's condition.

Administration of Lubricin

Generally, a therapeutically effective amount of lubricin foradministration systemically is in the range of 0.1 mg/kg to 100 mg/kg,or 1 mg/kg to 100 mg/kg, or 1 mg/kg to 10 mg/kg. In one embodiment, thedose of lubricin is between 0.25 mg/kg and 2.5 mg/kg or is between 0.25and 3.0 mg/kg. The amount administered will depend on variables such asthe type and extent of the condition to be treated, the overall healthof the patient, the pharmaceutical formulation, and the route ofadministration. The initial dosage can be increased beyond the upperlevel in order to rapidly achieve the desired blood-level or tissuelevel. Alternatively, the initial dosage can be smaller than theoptimum, and the dosage may be progressively increased during the courseof treatment. The optimal dose can be determined by routineexperimentation. For parenteral administration a dose between 0.1 mg/kgand 100 mg/kg, alternatively between 0.5 mg/kg and 50 mg/kg,alternatively, between 1 mg/kg and 25 mg/kg, alternatively between 2mg/kg and 10 mg/kg, alternatively between 5 mg/kg and 10 mg/kg isadministered and may be given, for example, once weekly, once everyother week, once every third week, or once monthly per treatment cycle.

For administration, lubricin is preferably combined with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means buffers, carriers, and excipients suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. Thecarrier(s) should be “acceptable” in the sense of being compatible withthe other ingredients of the formulations and not deleterious to therecipient. Pharmaceutically acceptable carriers include buffers,solvents, dispersion media, coatings, isotonic and absorption delayingagents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art. Useful formulations can beprepared by methods well known in the pharmaceutical art. For example,see Remington's Pharmaceutical Sciences, 18th ed. (Mack PublishingCompany, 1990). Formulation components suitable for parenteraladministration include a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as EDTA; buffers such as acetates,citrates or phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose.

Lubricin for administration can be presented in a dosage unit form andcan be prepared by any suitable method and should be formulated to becompatible with its intended route of administration. Examples of routesof administration are oral, intravenous (IV), intradermal, subcutaneous,intramuscular, inhalation, transdermal, topical, transmucosal, rectaladministration, parenteral, intranasal, topical, oral, or localadministration, such as by a transdermal means, for therapeutictreatment. Additional routes of administration include intravascular,intra-arterial, intratumor, intraperitoneal, intraventricular,intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital,rectal, topical, or aerosol inhalation administration.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol), and suitable mixturesthereof.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims. These and many other variations and embodiments of the inventionwill be apparent to one of skill in the art upon a review of thedescription and examples.

1. A method of reducing or inhibiting extravasation of cells from thecirculatory system of a patient in need thereof comprising administeringby injection directly or indirectly to the vascular system a compositioncomprising a physiologically compatible vehicle and PRG4 in an amountsufficient to contact PRG4 with an activated epithelial cell surface ofthe vasculature to produce a PRG4 film associated with at least aportion of the surface thereby to present a boundary lubricant layereffective at reducing or inhibiting the process of extravasation.
 2. Themethod of inhibiting or reducing intravasation of cells from anextravascular compartment to the circulatory system of a patientcomprising administering by injection directly or indirectly to theextravascular compartment a composition comprising a physiologicallycompatible vehicle and PRG4 in an amount sufficient to contact PRG4 withthe cells or cell-surrounding matrix to produce a PRG4 film associatedwith the surface or the matrix thereby to present an boundary lubricantlayer effective at reducing or inhibiting the native process of cellmotility or intravasation.
 3. The method of claim 1, wherein the cellsare neoplastic cells or leukocytes.
 4. The method of claim 1, whereinthe cells are cancer cells.
 5. The method of claim 4, wherein the cancercells are prostate cancer cells, breast cancer cells, lung cancer cells,or ovarian cancer cells.
 6. The method of claim 1, wherein the PRG4 isadministered to provide a dose of between 0.3 mg/kg and 3.0 mg/kg ofPRG4 in said patient.
 7. The method of claim 1, wherein the injection isa systemic intravenous injection or a localized injection at the site ofthe cells.
 8. The method of claim 1, wherein the extravasation of cellsreduced or inhibited is metastasis of cancer cells to bone, lymph,lungs, brain, or liver.
 9. The method of claim 4, wherein administrationof PRG4 occurs in conjunction with surgical, chemotherapy, or radiationtreatment for the cancer. 10-40. (canceled)
 41. The method of claim 2,wherein the cells are neoplastic cells or leukocytes.
 42. The method ofclaim 2, wherein the cells are cancer cells.
 43. The method of claim 42,wherein the cancer cells are prostate cancer cells, breast cancer cells,lung cancer cells, or ovarian cancer cells.
 44. The method of claim 2,wherein the PRG4 is administered to provide a dose of between 0.3 mg/kgand 3.0 mg/kg of PRG4 in said patient.
 45. The method of claim 2,wherein the injection is a systemic intravenous injection or a localizedinjection at the site of the cells.
 46. The method of claim 2, whereinthe extravasation of cells reduced or inhibited is metastasis of cancercells to bone, lymph, lungs, brain, or liver.
 47. The method of claim46, wherein administration of PRG4 occurs in conjunction with surgical,chemotherapy, or radiation treatment for the cancer.